LRP-1 receptor combines EGFR signalling and eHsp90α autocrine to support constitutive breast cancer cell motility in absence of blood supply

Tumor cells face constant stress of ischemic (nutrient paucity and hypoxia) environment when they migrate and invade too fast to outgrow the nearest blood vessels. During the temporary loss of support from circulation, the tumor cells must act self-sufficient to survive and then to migrate to re-connect with the nearest blood supply or die. We have previously reported that ablation of the low-density lipoprotein receptor-related protein 1 (LRP-1) completely nullified the ability of tumour cells to migrate and invade under serum-free conditions in vitro and to form tumours in vivo. The mechanism behind the important function by cell surface LRP-1 was not fully understood. Herein we show that LRP-1 orchestrates two parallel cell surface signalling pathways to support the full constitutive tumour cell migration. First, LRP-1 stabilizes activated epidermal growth factor receptor (EGFR) to contribute half of the pro-motility signalling. Second, LRP-1 mediates secreted Hsp90α autocrine signalling to bring the other half of pro-motility signalling. Only combined inhibitions of the EGFR signalling and the eHsp90α autocrine signalling led to the full blockade of the tumour cell migration as the LRP-1 depletion did. This finding uncovers a novel mechanism by which certain breast cancer cells use LRP-1 to engage parallel signalling pathways to move when they lose contact with blood support.


Results
Self-supported breast cancer cell motility versus serum-dependent breast epithelial cell motility. Constitutive motility even in the absence of blood supply is a critical ability for tumour cells to achieve continued invasion and metastasis 15 . To investigate the mechanism of this intrinsic cancer cell property, we first defined the so-called "self-supported and constitutive motility" of tumour cells by comparing the highly malignant human breast cancer cell line, MDA-MB-231 (https:// www. atcc. org/ produ cts/ htb-26), with the nontumorigenic human epithelial cell line, HBL-100 (https:// lincs. hms. harva rd. edu/ db/ cells/ 51117/). We utilized the colloidal gold migration assay, arguably the most sensitive and accurate cell migration assay that detects and quantitates single cell motility under any given condition by a darkfield microscope with assistance from a computer software, giving rise to a statistical sum of the so-called "migration Index" (%) of ("Materials and methods") [16][17][18] . As shown in Fig. 1A, each migration track (black area) represents the migration trail of a single and serum-starved (16 h) cell under indicated environmental conditions. For example, in the absence of serum factor support, the non-cancer HBL-100 cells were entirely unable to migrate (panel b, indicated by white dotted circle). In contrast, exactly the same and a duplicate set of the cells migrated to create large tracks only when 10% FBS was added to the medium (panel a). Under the full support of serum factors, MDA-MB-231 cells also migrated to create large tracks (panel c), as expected. However, the detected motility from MDA-MB-231 cells was largely self-supported and serum-independent, since it was only slightly reduced under serum-free conditions (emphasized by red dotted circle, panel d). The contribution of hypoxia (1% O 2 ) to cell motility was limited to both normal (panels e,f) and tumour (panels g,h) cells, where cell migration remained largely unchanged, in comparison to the effect of serum-free versus serum. Therefore, we mainly focus on serum-free conditions to mimic the lack of blood supply to the tumour cells in vivo for rest of the experiments, unless it becomes necessary controls. Computer-assisted quantitation of randomly selected 15 independent images of the cell migration on collagen-coated colloidal gold surface under each of the indicated conditions is shown in Fig. 1B. The data clearly indicate that MDA-MB-231 cells have acquired a self-supported motility under ischemia-like conditions. The lack of constitutive migration of HBL-100 cells under serum-free conditions was not due to a cell health issue, since crystal violet staining showed not only viability of the cells at the start (day 1, panels i,k) and even an increased cell number at the end of the experiment (day 2, panels j,l) under either normoxia or hypoxia (Fig. 1C). For rest of experiments, we use "231 cells" as the abbreviation of MDA-MB-231 cells.
Presence of LRP-1 supports the full constitutive tumour cell motility. The previous findings by many laboratories on LRP-1's support for tumour cell migration, invasion and tumour formation 7,8,[11][12][13][14] led us to investigate the role of LRP-1 in maintaining the constitutive motility and the possible mechanism in 231 cells, as schematically depicted in Fig. 2A. We used lentiviral infection-mediated shRNA delivery to downregulate LRP-1. The efficiency of LRP-1 down-regulation in the cells is shown in Fig. 2B (panel a, lane 2 vs. lane 1) and the specificity or possible off-target concern for the shRNA-LRP-1 is addressed in a later section (see Fig. 5). When the LRP-1-downregulated 231 cells were subjected to colloidal gold migration assay, as shown in Fig. 2C, we found that 1) the LRP-1-depleted cells have completely lost self-supported motility under serum-free conditions (panel e vs. panel d) and 2) LRP-1 downregulation did not affect physiological stimulus-, i. e. serum-stimulated motility of the same cells (panel f). We confirmed this important finding using an independent cell migration assay, the "scratch" assay, which measures the relative and directed migration of a cell population. As shown in Fig. 2D, under serum-free conditions, migration of the 231-LRP-1-KD cells was drastically delayed (panels i and  www.nature.com/scientificreports/ j), in comparison to 231-wt cells (panels g, h). It must be point out that a basal level of cell migration in "scratch" assay, instead of zero migration, was due to an intrinsic issue of this assay: natural diffusion by the crowded cell monolayer toward an open cell-free area. Computer-assisted quantitation of both motility assays is shown in Fig. 2E,F. Results of the quantitation demonstrated that the LRP-1 plays a critical role in self-supported motility of the tumour cells.
The "eHsp90α > LRP-1 autocrine loop" only accounts for a portion of the self-supported tumour cell motility. To investigate the possible mechanism by which the LRP-1 receptor supports the constitutive motility of the tumour cells, we initially speculated that the previously identified "tumour-secreted Hsp90α > LRP-1 receptor" autocrine pathway 19,20 as schematically shown in Fig. 3A, must be the candidate. To study this hypothesis, we focused on the serum-free conditioned medium (CM) of the 231-wt and 231-Hsp90α-KO (231α-KO) cells. Total lysates of two independent 231α-KO clones, α-KO-#1 and α-KO-#2, were blotted with anti-Hsp90α antibody (Fig. 3B, panel a), which we have previously generated using CRISPR- Human recombinant (hr) Hsp90α protein-stimulated migration was included as the positive control (panel j). These results suggest that secreted Hsp90α in the CM of 231-wt cells was a main stimulus of target cell migration. Computer-assisted quantitation of the migration data is shown Fig. 3G, clearly showing the critical role for secreted Hsp90α. However, to our total surprise, neither depletion of Hsp90α nor mAb 1G6-D7 neutralization of the secreted Hsp90α function from the extracellular environment could replicate the full effect of LRP-1 downregulation. As shown in Fig. 3H, in comparison to LRP-1 down-regulation (panel n), neither Hsp90α-KO, α-KO-#1 cell, (panel o) nor anti-Hsp90α neutralizing antibody, mAb1G6-D7, (panel p) was able to achieve a complete inhibition of the self-supported motility of the tumour cells. Similar results were obtained from an independent Hsp90α-KO cell clone, α-KO-#2, (panels q to t). The finding of two independent cell clones and mAb1G6-D7 was confirmed by data quantitation (Fig. 3L).
To further substantiate the finding of the critical role for secreted Hsp90α instead of other factors in the CM, as shown in Fig. 4A, we found that CM from either 231-wt (panel d) or 231-α-KO (panel e), as well as hrHsp90α protein (panel c), were all unable to promote migration of the 231-LRP-1-KD cell, whereas the cells still achieved full migration under serum stimulation (panel b). Computer-assisted quantitation of these migration data is shown in Fig. 4B. Finally, on the 231-LRP-1-KD cells, we found that hypoxia showed little rescuing effect. As shown in Fig. 4C, LRP-1 downregulation blocked 231 cell migration (panel h vs. f). Hypoxia had little effect on the results (panels j and l), whereas the cells responded fully to serum as expected (panels g vs. i and panels k vs. m). Quantitation of these data shown is presented in Fig. 4D, which indicates that hypoxia's specific contribution to tumour cell constitutive motility is limited. Taken together, the above data indicate that i) secreted Hsp90α is LRP-1 stabilizes activated EGFR that accounts for the second pro-motility pathway. The previous finding suggested that, besides the "secreted Hsp90α > LRP-1 receptor" autocrine pathway, LRP-1 regulates another independent signalling pathway to achieve a full control of the self-supported tumour cell motility. To identify the second pathway, we carried out antibody screening for stability of the major signalling molecules in the LRP-1-downregulated 231 cells and made a surprising finding. As shown  To this argument, we present here the 19-nucleotide shRNA sequence, designed by the Synthego CRISPR Design Tool (https:// www. synth ego. com/ produ cts/ bioin forma tics/ crispr-design-tool) that minimizes cross reactivity of an shRNA, in alignment with the human EGFR cDNA that encodes shRNA-targeting EGFR mRNA. First, as shown in Fig. 5C, there are 12 continuous nucleotide sequence (in green) from human EGFR gene that matched 12 of the 19-nucleatide shRNA against human LRP-1 gene. Second, nonetheless we wanted to provide direct evidence for our findings that i) our shRNA against LRP-1 does not cross react with EGFR and ii) LRP-1 downregulation selective weakens the activated EGFR stability. To achieve the two goals at once, we took the advantage of primary human dermal fibroblast (HDF) cells that express inactive EGFR under serum-free conditions and exhibit EGFstimulated tyrosine-phosphorylation of EGFR in a time-dependent fashion. Thus, we compared the stability of the EGFR between parental and LRP-1 downregulated HDFs without or with EGFR stimulation over time. As shown in Fig. 5D, the shRNA-mediated downregulation of LRP-1 was near complete (panel l, lanes 5-8 vs. lanes 1-4), with quantitation of the band intensities in reference to the β-actin's by Image J shown underneath. Under these conditions, it is clearly shown that there was little cross reactivity of the shRNA against EGFR (panel m, lane 5 vs. lane 1, with an asterisk mark in red), instead a slight increase in EGFR. However, the EGF-activated EGFR declined significantly faster in LRP-1-KD cells (lanes 6-8) than the parental cells (lanes 2-4) with at least a three-fold difference based on the Image J data. The Hsp90 (panel n) and β-actin (panel o, the basis for Image J) were included as controls.
Simultaneous inhibitions of both eHsp90 > LRP-1 and EGFR signalling pathways completely eliminate the self-supported motility of the cancer cell. We speculated that LRP-1 is at the pivotal position to coordinate both the "eHsp90α > LRP-1" autocrine signalling and the EGFR signalling to support the constitutive motility of the cancer cells under serum-free conditions. First, we tested the role of the activated EGFR in the self-supported motility of 231 cells by using the EGFR tyrosine kinase-specific inhibitor, Gefitinib (C 22 H 24 ClFN 4 O 3 ). As shown in Fig. 6A, while most previous studies recommended 5 μM or higher concentrations of Gefitinib, we chose 3 μM as our working concentration, which dramatically reduced the phos- Computerassisted quantitation of the migration data is shown in Fig. 6E, which clearly indicate that the tumour cells utilize LRP-1 to coordinate both eHsp90α signalling and EGFR signalling for maintaining self-supported motility under serum-starved conditions.

Discussion
Tumour cell self-supported motility serves as a foundation for tumour invasion and metastasis, which kill patients. Similar to the findings that different oncogenes and tumour suppressor mutant genes drive tumorigenesis of different types of tumours, the mechanisms behind tumour cell self-supported motility likely vary among different tumours and currently remain largely understudied. Understanding the mechanisms could guide the designs of target-specific therapeutics or even personalized anti-tumour drugs. In the current study, we have taken the highly malignant and well-characterized human triple negative breast cancer cell lines, MDA-MB-231 (231), as the cell model to report the first mechanism that drives self-supported tumour cell motility under serum-free conditions, a characteristic microenvironment during tumorigenesis. In contrast to the conventional wisdom of mutated oncogene-driven mechanism, our study has revealed a parallel dual signalling pathwayparticipated mechanism with the cell surface LRP-1 as the central coordinator. As schematically depicted in Fig. 7, LRP-1 receptor maintains stability of tyrosine-phosphorylated EGFR signalling at one hand and mediates the tumour cell-secreted eHsp90α autocrine signalling on the other hand, to achieve a full self-supported motility that reaches about 60% of the full cell motility under physiological conditions. We suggest that in the absence of sufficient serum support, the "starved" tumour cells constantly depend upon this self-supported migration to move away from the (temporal) hazard environment of no blood support until they gain new environmental support. The finding that LRP-1 maintains the stability of activated EGFR cells is not cell type-specific. Similarly, another highly related breast cancer cell line, MDA-MB-468, has an undetectable level of LRP-1 and yet a much higher level of inactive EGFR 7 . Moreover, EGFR in primary human dermal fibroblasts under serum starvation remains inactivate, unless EGF is added to the medium. We showed that down-regulation of LRP-1 had little effect on the inactivated EGFR in the absence of EGF stimulation. However, the level of EGF-stimulated EGFR declined dramatically faster in LRP-1-downregulted cells (see Fig. 5D). Currently, how LRP-1 maintains the stability of active EGFR remains unknown. Since the full-length cDNA for LRP-1, which encodes for a 515-kDa extracellular α subunit and an 85-kDa trans-membrane β subunit, exceeds the maximum cloning limit for any cDNA expression vectors 21 , it is technically not possible to do rescue experiments with the full-length LRP-1 cDNA. Our preliminary study show that none of the four mini-LRP-1 receptor cDNAs that encodes the transmembrane 85-kDa β subunit plus one of the four extracellular domains (I to IV) 22 was able to stop the faster downregulation of EGF-stimulated EGFR. These results suggest that combined extracellular domains or even the full-length LRP-1 is required for stabilizing activated EGFR at cell surface. A more exciting possibility is what www.nature.com/scientificreports/ role for LRP-1 to play in stability of tyrosine kinase receptors in general. So far, Takayama et al. reported that LRP1 controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor β (PDGFRβ) 23 . Gopal and colleagues showed that the eHsp90-LRP1 signalling axis involves activation of the EphA2 tyrosine kinase receptor 14 . Together with our current finding of LRP-1 on active EGFR, it is of a great interest to further explore the broader possibility that LRP-1 serves as a "mother co-receptor" for all tyrosine kinase receptors. Independent of the EGFR signalling, the second parallel "eHsp90α > LRP-1 autocrine loop" to promote tumour cell migration has been clearly established by previous studies 8,13,19,22 . Normal cells secrete eHsp90α only under stress, such as tissue injury signals, whereas many tumour cells have acquired the ability to constitutively secrete Hsp90α driven by oncogenes, such as overexpressed HIF-1α and mutant p53 8,24,25 . The sole function of eHsp90α is to promote cell motility during wound healing, which is taken advantage of by tumour cell invasion. Secreted Hsp90α acts as a bona fide signalling protein that binds to the subdomain II in the extracellular part of LRP-1 and activates, via the NPVY motif in the cytoplasmic tail of LRP-1, the Akt and Erk1/2 kinases 22 . In all, this current study is the first report on a novel mechanism that supports tumour cells' constitutive motility, a prerequisite for tumour invasion and metastasis.

Lentivirus production, infection and Western blot analysis.
The selected shRNA sequence (sense) against human LRP-1/CD91, GAC CAG TGC TCT CTG AAT A, was cloned into lentiviral vector, FG12, for delivering shRNA was as previously described 8,19 . This construct was mixed with two packaging vectors, pCMVΔR8.2 and pMDG, and used to transfect 293 T cells to produce virus stocks for infection of target cells, such as 231 and HDF cells. Following infection, total cell lysates of different treatments were equalized using BCA Protein Assay Kit (Thermo Scientific). Protein samples were resolved in SDS-PAGE, transferred onto a nitrocellulose membrane and stained with Ponceau S solution to confirm efficient and even protein transfer. The membrane was blocked by 5% BSA (in TBS buffer) prior to incubation with primary antibodies against as indicated. Secondary anti-rabbit IgG (1:10,000) and anti-mouse IgG (1:10,000) were used as instructed by manufacturers, followed by ECL reaction. Protein bands from the same blot of the same experiment was quantitated using the NIH Image J software 7,22 . Crystal violet staining of live cells. 50% confluence cells were serum starved for one day or two days, respectively according to the cell migration protocols. Cells were rinsed with PBS three times to remove dead cells and the attached cells were stained with 0.4% crystal violet solution in 20% methanol for 10 min at room temperature. The plates were washed three times with water and inverted on filter paper for air dry at room temperature. Adherent cells were imaged at 10 × magnification using a light microscope. Randomly selected 5 images (2 mm × 1.4 mm each) for each condition under the microscope were recorded. Cell number within 0.3mm 2 was counted for each recorded image. The cell numbers from the 5 images per condition were averaged to reflect the cell survival rate.
Cell survival and growth assay. Cells during their exponential growth phase were used for this assay.
Cells were re-seeded at 8 × 10 4 cells/well in 12-well plate. The attached cells in triplicates were lifted by trypsin and cell number counted as day 0 start point (without treatments). The medium was replaced with complete medium without or with indicated concentrations of Gefitinib and incubated for 48 h. The number of viable cells in each well was counted and averaged numbers from triplicates per concentration plotted as number of cells versus increasing concentrations of Gefitinib. All numerical results are reported as mean and standard deviation (s.d.).
Colloidal gold cell motility assay and "scratch" cell migration assay. Cell motility colloidal gold phagokinetic assay with computer-assisted quantitation was as previously invented by Albrecht-Buehler and Scientific Reports | (2022) 12:12006 | https://doi.org/10.1038/s41598-022-16161-y www.nature.com/scientificreports/ modified by our laboratory 16,17 . Briefly, bovine serum albumin (BSA, 1%) -coated coverslips (35 mm in diameter) were placed into 12-well tissue culture plates with one coverslip per well. Gold salt solution (9% of gold salt is combined with 52% of H2O and 30% of the Na2CO3 solution) was heated in a 50 ml Erlenmeyer flask with constant swirling until boiling, removed from the heat source. An equal volume of freshly prepared formaldehyde (0.1%) was slowly added to the gold salt mixture with gentle swirling. After the mixture turned brown, the solution was immediately pipetted into the 12-well plates with BSA-coated coverslips at 1 ml per well. The plates were covered and left undisturbed for at least 2 h to settle gold salt particles. Following removal of the upper liquid solution, the coverslips were gently rinsed once with 1 ml of Hank's buffered salt solution (HBSS) and coated with type I collagen in HBSS at 37 °C for 2 h. After free collagen was removed and plates were rinsed once with HBSS, 3,000 cells were plated into onto each well with indicated conditions and allowed to migrate for 16 h. Cell migration was examined under dark field microscope linked to a computer to visualize on screen. Fifteen randomly selected and nonoverlapping fields under each experimental condition were analyzed with an attached CCD camera (Model KP-MIU, Hitachi-Denshi) and a computer using the NIH Image 1.6 program, which calculates migration index (MI), which represents the percentage (%) of the cell migration tracks-consumed area over the total field area viewed by the microscope. A modified procedure of the in vitro wound-healing ("scratch") assay was performed according to Li and colleagues 17 . The main modifications include: (1) coating the surface of the tissue culture wells with a specified ECM, e. g. collagen, fibronectin and (2) the wounds/scratches are made shortly after the cells fully attached (within 2-3 h following seeding) and floating cells removed. The pre-coated ECM and the relatively short cell attachment period prevented additional ECM deposition on the migratory surface contributed by the cells themselves. Mitomycin C (10 μg/ml) was included throughout the experiments to prevent cell proliferation. Each condition has triplicate wells for quantitation. Quantitation was made based on the (left) open cell-free area, in reference to 100% on day 0. Statistical analysis. Statistical significance was determined using a two-tailed Student's t-test and one-way ANOVA. Final presentation as mean ± s.d. was based on at least three independent and corroborating experiments. Confirmation of a difference in migration as statistically significant requires rejection of the null hypothesis of no difference between mean migration indices obtained from replicate sets. The p value equal or less than 0.05 was considered statistically significant.